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LNA Oligos are composed of either 100% Lock Nucleic Acid (LNA) bases or a mixture of LNA and DNA bases. However, only oliognulceotides that are between 7 and 15 mers in length can be made up of 100% LNA. If an oligonucleotide is longer, the 100% LNA oligo may bind to itself (self complement) rather than to the target sequence. Therefore, for oligonucleotides longer than 15 mers, we recommend decreasing the LNA content as the oligonucleotide increases in length, as follows:
If longer sequence is absolutely necessary, we can synthesize client's existing design as chimera with mixture of LNA bases and DNA bases and phosphorothioate modifications only the DNA gap, leaving LNA flanks phosphodiester LNA base linkage. This is the only way we can synthesize longer sequences.
In vivo Knockdown
For in vivo knockdown, consider using complete phosphorothioate backbones, and also consider conjugated cholesterol oligos or cell penetrating peptide for better uptakes in the cell.
Design Guidelines for SNP Microarray Genotyping
Guidelines for designing LNA containing oligonucleotides for genotyping using SNP chip microarrays are listed below. Please note that all the general design guidelines also apply. the guidelines should be considered as a rule of thumb.
Capture probes should be approximately 12 bp in length.
2-3 LNA bases should be positioned directly at the SNP site.
The position of the mismatch in the capture probe is flexible - however, positioning the SNP at the very 3' or 5' end or 1 position from the ends may compromise discrimination.
A Tm of approximately 65 oC is recommended.
No LNA bases should be positioned in aplindrome sequences (GC base pairs are not allowed, while AT base pairs are less critical).
Design Guidelines for PCR Primers
LNA's should be introduced at the positions where specificity and discrimination is needed (e.g. 3' end in allele specific PCR and in the SNP position in allele specific hybridization probes).
Avoid stretches of more than 4 LNA bases. LNA hybridized very tightly when several consecutive residues are substituted with LNA bases.
Start by spiking LNA in the 5' end of the primer (allow the 5' end of the primer to anneal at high TM avoiding random priming by un- specific annealing of the 3' end).
Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
Typical primer length of 18mer should not contain more than 8 LNA bases.
Each LNA bases increases the Tm by approximately 2-4 oC.
Do not use blocks of LNA near the 3' end.
Keep the GC-content between 30-60%
Avoid stretches of more than 3 G DNA or LNA bases.
Tm of the primer pairs should be nearly equal.
Design guidelines for Allele Specific PCR
For improvement of allele specific PCR a single LNA nucleotide should be placed in the terminal 3' or the 3'-1 position. In both cases the LNA base should correspond to the position of the polymorphism. Follow general design guidelines above.
Design guidelines for Real Time qPCR Probes
The 3'-end of the labeled probe should be blocked with PO4, NH2 or a blocked nucleotide to prevent extension.
Tm of the labeled probe should optimally be 10 oC higher that Tm of the forward primer. For single mutation detection the Tm-difference should be 7 oC
Typical Tm of PCR primers for labeled assays: 58-60 oC
Typical Tm of labeled probes: 65-70 oC (i.e. slightly lower than the extension temperature).
Optimal length of LNA substituted labeled probes: 15-18 nucleotides (Please note that these are 5-8 bases shorter than the corresponding DNA probes).
Maintain Tm with LNA substitutions to match the Tm of the corresponding longer DNA probes.
Substitute every third base with LNA in the central segment of the probe. Usually 4-6 LNA substitutions are required to obtain a useful Tm.
When detecting single nucleotide mutations, select the probe sequence so thtat the mutation is located centrally in the probe. Make a single LNA substitution at the position of the single nucleotide mutation.
Avoid LNA substitutions participating in formation of secondary structures.
Position the labeled probe as close as possible to the forward primer.
Avoid Guanine (G) in the 5'-position next to the fluorophore.
Select the strand giving the lowest concentration of G's in the probe.
Avoid longer stretches of identical nucleotides and especially G's.
All fluorescent dyes offered by Bio-Synthesis can be conjugated to LNA containing probes. (FAM, TET, HEX, TAMRA, ROX, CY3, CY3.5, Texas Red, Cy5, Cy5.5, Cy7 and Alexa series dyes and more...)
LNA (Locked Nucleic Acids) is a registered trademark of Exiqon A/S. Visit design and analysis tools provided by Exiqon or save time by letting Bio-Synthesis design your LNA probe or primers by sending us your design query here.
Visit our LNA reference literature download site.
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